MR extracellular volume mapping and non-contrast T1ρ mapping allow early detection of myocardial fibrosis in diabetic monkeys

Eighteen rhesus monkeys with spontaneous T2DM (11 to 19 years old, with 1- to 5-year duration of T2DM) and nine healthy control (HC) rhesus monkeys matched for gender, age, and weight were studied. The criteria used for rhesus monkey selection were guided by our previous work [17] and required a fasting plasma glucose (FPG) level of 5.5 mmol/L or more and a glycated hemoglobin (HbA1c) level of 4.5% or more [16, 18]. None of the monkeys had a history of medical treatment. All rhesus monkeys used in this study were provided by Sichuan Primed Bio-Tech Co., Ltd. (Sichuan, China). The monkeys were maintained in accordance with the requirements of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Association for Assessment and Accreditation of Laboratory Animal Care. All experimental protocols were reviewed and approved by the Experimental Animal Ethics Committee of West China Hospital, Sichuan University, Chengdu 610041(Institutional approved number 2015065A) and by the Institutional Animal Care and Use Committee of Sichuan Primed Group Co., Ltd.

The general condition and related blood biochemical indicators of all monkeys are shown in Table 1. Information about animal preparation before echocardiographic and CMR examination can be found in the supplements.

Transthoracic echocardiography was performed using a standard protocol [19] with the GE Vivid S5 hand-carried Doppler system (GE Medical Systems, Israel Ltd.). Various diastolic function indicators were all measured, including the mitral early diastolic peak (E-wave) and the late peak (A-wave) velocities, the E/A ratio, the early diastolic mitral annulus peak velocity (E’), the late diastolic mitral annulus peak velocity (A’), the E/E’ ratio, and the E’/A’ ratio. Data were averaged across three cardiac cycles.

In accordance with the classification of DD in our previous study and in humans [20, 21], monkeys were divided into three DD severity groups for data analysis. Monkeys with normal diastolic function (i.e., E/A > 1, E’/A’ > 1, E’ > 7, and E/E’ < 11) were assigned to group 1. Monkeys that show E/A < 1, E’/A’ > or < 1, E’ > 7, and E/E’ < 11 were classified as having mild DD and assigned to group 2, and monkeys that show E/A > 1, E’/A’ < 1, E’ < 7, and E/E’ > 11 were classified as having moderate DD and assigned to group 3.

CMR imaging was performed using a 3.0 Tesla magnetic resonance imaging system (Magnetom Trio; Siemens Medical Systems) with a 32-channel cardiac surface coil (Siemens).

A CMR cine sequence was performed to scan 10 to 14 short-axis slices via a steady-state free precession with retrospective electrocardiogram triggering. Next, the T1ρ images were obtained using an electrocardiogram-gated T1ρ-prepared gradient echo sequence in three short-axis slices at the base, middle, and apex of the left ventricle. A modified look-locker inversion recovery (MOLLI) sequence with motion correction was then applied to obtain pre-contrast T1 maps with an acquisition scheme of 5(3)3 [22, 23]. Ten minutes after contrast injection (0.15 mmol/kg of Magnevist; Bayer Health Care Pharmaceuticals), post-contrast MOLLI T1 mapping was acquired with a scheme of 4(1)3(1) [22, 23] in the same three short-axis slices that were used for the T1ρ scans. Late gadolinium enhancement images were obtained using an electrocardiogram-gated breath-hold inversion recovery Turbo FLASH at 15 min after contrast injection (Table 2).

All CMR images were prospectively analyzed. A blood sample was taken from all monkeys immediately before each CMR study for hematocrit measurements. Cine and ECV mapping were analyzed offline with cmr⁴²© software (Circle Cardiovascular Imaging Inc.). The following parameters were obtained: left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), stroke volume (SV), left ventricular ejection fraction (LVEF), left ventricular (LV) mass, and average heart rate (a-HR). Based on the cine sequence, we also performed strain analysis using cmr⁴²©-based feature tracking. Both the global peak systolic longitudinal strain (GSL) and the global peak diastolic longitudinal strain rate (GSrL) were obtained. Based on the pre-contrast and post-contrast T1 maps, which were automatically generated with a prototype inline process function from Siemens, ECV maps were generated by cmr⁴²©. If there was motion between the pre- and post-T1 series, the pre- and post-MOLLI were registered using a cmr42© intensity-based registration method then ECV maps were calculated. The myocardial fibrosis index (mFI) was calculated as follows:where the spin-locking frequency (SLF) is ω₁ = γB₁ and γ is the gyromagnetic ratio. The T1_ρ(0) value is relatively constant for myocardial tissue with different quantities of fibrosis. Although the mFI is calculated in millisecond, it was used as an index that had been calculated in arbitrary units for the purposes of this study. All of the image processing and analyses were performed with a custom-written software (ImPro_MR_Analysis_Suite), which was created in MATLAB (MathWorks). Segmental [24] ECV values, mFI values, and T1ρ values were all acquired. Whole-heart average values were obtained, with the exclusion of artifact segments. Since mFI represents the subtraction of two T1ρ images, the artifacts exclusion of T1ρ and mFI is based on the original T1ρ-weighted images. Signal intensity values of more than the mean plus 5 multiplied by the standard deviation of the signal intensity in remote normal myocardium were used to determine regional fibrosis.

One diabetic monkey was given deep anesthesia (pentobarbital sodium, P3761; Sigma-Aldrich) to euthanasia. Histologic slices from the anterior interventricular septum were excised. After dehydration and embedding, the slices were subjected to the Masson staining protocol and analyzed under an optical microscope (BX43F; Olympus).

Statistical analysis was performed using SPSS version 22 (IBM). All data were checked for normality using the Shapiro-Wilk test and presented as the mean ± standard deviation and mean (95% confidence interval (CI)) or as a median (Q1–Q3), as appropriate. Normally distributed data sets were analyzed with one-way analysis of variance, and Fisher’s least significant difference test and Student-Newman-Keuls test were used in post hoc analysis. The Kruskal-Wallis H test was used to analyze non-normal distribution; the Dunn-Bonferroni test was used in post hoc analysis. Bivariate correlations were performed using either the Pearson or Spearman method, as appropriate. Significance was assumed at p < 0.05.

There were nine T2DM monkeys with mild DD in group 2 and nine T2DM monkeys with moderate DD in group 3. The nine HC monkeys in group 1 demonstrated normal DD (Supplemental Table 1).

The baseline CMR results summarized in Table 3 show that there were no significant differences in LVESV, LVEDV, SV, LVEF, LV mass, and a-HR among the three groups. Despite the non-significant findings related to the basic cardiac function of the three groups, the global myocardial ECV (%) was significantly different among the three groups (24.61 (22.96–25.88) vs. 26.34 (25.39–26.97) vs. 29.02 (27.46–34.48); p < 0.000), and the ECV values of group 3 were significantly higher as compared with those of group 2 (p = 0.027). However, there was no significant difference in ECV values between group 1 and group 2 (p = 0.307) (Figs. 1 and 2). There was no evidence of regional late contrast enhancement in any of the monkeys. On the other hand, T1ρ relaxation time was significantly higher in groups 2 and 3 as compared with group 1; however, there was no significant difference between group 2 and group 3. The mFI increased progressively from healthy animals to those with mild DD and then to those with moderate DD (2.90 (1.82–4.00) vs. 4.91 (3.30–6.51) vs. 7.74 (5.84–9.65); p < 0.000). The differences between group 1 and group 2 (p = 0.049) and between group 2 and group 3 (p = 0.007) were all significant. GSrL values in group 3 were significantly lower than those of group 1 (p = 0.013). The results of the post hoc analysis are shown in Supplemental Table 2.

There was a moderate positive correlation between ECV and mFI (r = 0.603) (Fig. 3). A positive correlation between ECV and T1ρ relaxation time was also found (r = 0.582).

GSrL had a negative correlation with mFI (r = − 0.421) and ECV (r = − 0.607). GSL was found to be correlated with ECV (r = − 0.434). E’ was correlated with mFI (r = − 0.465), T1ρ (r = − 0.549), and ECV (r = − 0.715) (Table 4; Fig. 4).

Perivascular and interstitial fibrosis were confirmed in this T2DM monkey with moderate DD with the use of Masson staining (Fig. 5). Collagen fibers were stained with aniline blue, and cardiomyocytes were stained red. The corresponding CMR ECV map and mFI map are also shown in Fig. 5. The average values of ECV and mFI in the approximate areas from which the histopathological slides were taken were found to be elevated (ECV = 37.46%; mFI = 5.85).

Due to the use of this rare, naturally occurring, chronic, NHP disease model of monkeys with T2DM as well as the longitudinal long-term study consideration, only one monkey with T2DM and DD was sacrificed for histopathology to show diffuse myocardial fibrosis. The ECV measurements in this study had not previously been validated in a monkey model. Given the close agreement of ECV among various species [39, 40], it is reasonable to assume that the ECV measurement in a monkey is still valid, although direct rigorous validation remains to be performed by histopathology. While we did monitor blood pressures immediately before and after CMR study, we did not have pressure data during the CMR study to normalize CMR results. This limitation will be resolved with the addition of MRI-compatible physiological monitoring at our institute [41]. Finally, although there are statistically significant differences in ECV and mFI between the different monkey groups, there is also an overlapping of values. Further studies with larger simple sizes are needed for vigorous validation beyond this initial investigation.